This invention relates to the germination of plant somatic embryos. More particularly, the invention relates to a process of treating plant somatic embryos with a nutripriming step to enhance subsequent germination of such embryos.
A plant seed is a complete self-contained reproductive unit generally consisting of a zygotic embryo, storage reserves of nutrients in structures referred to as cotyledons, endosperm or megagametophytes, and a protective seed coat encompassing the storage reserves and embryo. In nature, maturation of plant seeds is usually accompanied by gradual loss of water over a period of time to levels between 10-35% moisture content. Once these low moisture levels are achieved, plant seeds can be stored for extended periods.
Germination of zygotic plant seeds is generally triggered by one or more environmental cues such as the presence of water, oxygen, optimal temperature, and light. Seeds germinate by means of a series of events which commence with the uptake of water by a quiescent dry seed and then subsequently proceed through various biophysical, biochemical and physiological events which ultimately result in the elongation of the embryo along its axis.
For the purpose of simplifying discussion of the present invention, the continuous process of seed germination is divided into three phases. Phase one is referred to as imbibition and is characterized by a rapid initial influx of water into the seed. Other significant events occurring in Phase one are the initiation of repair to damage to DNA and mitochondria which may have occurred during seed desiccation and/or the maturation process, and subsequent commencement of protein synthesis facilitated by existing mRNA.
Phase two is characterized by a significant reduction in the rate of water uptake (i.e., imbibition has been completed). This is accompanied by activation or de novo synthesis of enzymes that specialize in hydrolyzing the complex storage reserves of carbohydrates, proteins, and lipids in the embryo and the cotyledons or megagametophytes. The hydrolysis of these complex storage reserves provides the substrates required for the respiration and growth of the zygotic embryos.
Phase three is characterized by a second rapid increase in the rate of water uptake. Water absorbed during Phase three is used primarily for the initiation of meristomatic cell division at the root and shoot apices of the embryo, and for uptake into the cells along the embryonal axis. Water taken up by the axial cells of the embryo applies turgor pressure which results in axial cell elongation. The net effect is that the embryo elongates to the point of protrusion through the seed coat. Protusion of a shoot or root radicle through the seed coat signifies the completion of germination and the onset of seedling growth and development.
The speed and success for germination of zygotic seeds can fluctuate considerably depending on various factors such as the residual influence of environmental conditions in which the seed developed and maturated, the amount of storage reserve compounds synthesized during the seed maturation process, the duration of storage, and the quality of the storage environment (e.g., temperature and humidity). From a commercial perspective, it is desirable to reduce the risk of germination failure and to ensure that seeds germinate rapidly and uniformly.
The commercial need for optimum seed germination performance has led to the development of processes known in the art for zygotic seeds as xe2x80x9cseed primingxe2x80x9d. This term may be defined as the xe2x80x9cuptake of water to initiate the early events of germination, but not sufficient to permit radicle protrusion, preferably followed by dryingxe2x80x9d (McDonald, 2000). Four techniques are currently used commercially to accomplish seed priming. These are hydropriming, osmopriming, matripriming and pregermination. However, regardless of the method used, the fundamental principles of seed priming are that: (1) the preliminary stages of germination are activated specifically and exclusively through controlling the availability of water to the seeds, and (2) the germination processes initated through an external priming process are subsequently arrested by a desiccation step.
One of the problems with commercializing somatic embryo technologies has been a relatively low rate of conversion to seedlings and low seedling vigor when conversion takes place. It would clearly be advantageous to improve such rates of conversion and levels of seedling vigor.
However, a significant additional problem is the current inability to use conventional horticultural ex vitro techniques, practices and environments for the sowing and germinating of plant somatic embryos. The main reason for the difficulties in successfully germinating plant somatic embryos in non-sterile commercial growing environments using conventional propagation practices is that during the intitial stages of germination, matured plant somatic embryos cannot produce their own carbon compounds or derive energy from photosynthesis. Furthermore, they lack the presence of their own energy and nutrient sources that are equivalent to storage reserves contained within cotyledons or endosperm or megagametophyte tissues in zygotic seeds. Consequently, an exogenous source of energy in the form of a selected sugar within a culture medium and other nutrients, must be supplied to the plant somatic embryos for successful germination to occur. Such culture media are highly susceptible to invasion by microorganisms which inevitably result in death or interfere with embryo survival and germination. Consequently, sowing and successful germination of plant somatic embryos on culture media must be conducted under strict aseptic conditions.
Although numerous protocols are known for the sowing and germination of somatic embryos and growing them into intact functional seedlings, all of these protocols are dependent on the use of aseptic techniques combined with in vitro systems that must be kept in biological isolation from contaminating microorgansisms and fungi until the plant somatic embryos have successfully completed germination and have achieved autotrophy. Consequently, none of these protocols has demonstrated compatibility with conventional horticultural equipment and practices.
Generally, the known protocols for germinating somatic embryos fall into two categories. The first is a category of protocols based on various in vitro methods which generally are comprised of sowing naked, i.e., uncoated, somatic embryos using aseptic techniques, onto sterilized semi-solid or liquid media contained within a solid-support such as a petri dish or a phytatray to facilitate germination under biologically isolated sterile conditions (e.g., U.S. Pat. Nos. 5,183,757; 5,294,549; 5,413,930; 5,464,769; 5,506,136 all of which are herein incorporated by reference) and subsequently, transplanting the germinants into conventional growing systems. The most significant disadvantages of such in vitro protocols for sowing naked somatic embryos are that (a) each embryo typically must be handled and manipulated by hand for the germination and transplanting steps, and (b) aseptic techniques and culture conditions must be rigorously maintained through to the step of transplanting of somatic germinants out of the in vitro germination media into horticultural growing media. Although various automation options, including robotics and machine vision, have been assessed for their usefulness in cost-effective reduction or elimination of the extensive hand-handling currently necessary to sow naked embryos (Roberts et al., 1995), no commercial equipment currently exists which can reliably, aseptically, and cost-effectively perform the in vitro protocols for germination of naked somatic embryos and subsequent transplanting into conventional propagation systems.
The second category of protocols teach encapsulation (generally gel-encapsulation) of somatic embryos (e.g., U.S. Pat. Nos. 4,777,762; 4,957,866; 5,183,757; 5,482,857 all of which are herein incorporated by reference) to provide a means by which the embryos can presumably be sown with mechanical devices such as seeders and fluidized drills, into conventional growing systems. However, there are a number of disadvantages with gel-encapsulated somatic embryos. For example, the hydrated semi-solid physical characteristics of encapsulated embryos make them incompatible for use with conventional seeding equipment currently available for commercial plant propagation, because the semi-solid gel-encapsulated somatic embryos tend to clump together during handling and consequently, are difficult to singulate and dispense. Furthermore, compositions of encapsulated embryos prepared as taught by the art, clog-up the conventional equipment, and for these reasons, it currently is not possible to sow encapsulated embryos with conventional seeding equipment. Consequently, novel equipment has been developed specifically for delivery of encapsulated somatic embryos into conventional growing systems. Such sowing devices have been reviewed by Sakamoto et al. (1995), but these devices have only been developed and tested as prototypes. Because of mechanical limitations and the high costs associated with the prototype mechanical seeders developed for sowing encapsulated embryos, none are currently available for commercial acquisition and use.
Another disadvantage with encapsulated somatic embryos is the lack of nutrient availability that is characteristically supplied to zygotic embryos by their attendant endosperm or megagametophyte tissues. Consequently, the encapsulation technology for somatic embryos has been extended to include the incorporation of various nutrients such as sugars, fertilizers, oxygen, into the encapsulation medium (e.g., Carlson and Hartle, 1995; U.S. Pat. Nos. 4,583,320; 5,010,685; 5,236,469, all of which are herein incorporated by reference). However, a distinct disadvantage associated with nutrient-amended encapsulated embryos is their susceptibility to microbial invasion during manufacture, storage, and during germination if germinated on non-sterile media.
Furthermore, it must be pointed out that although considerable prior art (e.g., PCT Patent Application WO 94/24847, and U.S. Pat. Nos. 5,010,685; 5,236,469; 5,427,593; 5,451,241; 5,486,218 all of which are herein incorporated by reference) teaches methods to manufacture xe2x80x9cartificial seedsxe2x80x9d consisting of somatic embryos encapsulated in gels, which may or may not be optionally supplemented with nutrients, and which may or may not be encased within a rigid covering, and although the prior art makes references to sowing said artificial seeds ex vitro into germination media comprised of soil or soil-less mixes, the prior art only teaches methods for germinating said artificial seeds in vitro, i.e., on sterilized semi-solid laboratory media. No methods are taught or otherwise disclosed in the prior art for sowing said encapsulated somatic embryos and/or manufactured and/or artificial seed into conventional growing systems using conventional sowing equipment.
However, the most significant disadvantage with all prior art procedures for encapsulating or otherwise coating somatic embryos, is that somatic embryos processed following those protocols typically have, as a consequence, much lower germination vigor and success than corresponding zygotic seeds (Carlson and Hartle, 1995). Carlson and Hartle (1995) concluded that considerable research is still required before xe2x80x9cmanufacturedxe2x80x9d or xe2x80x9cartificialxe2x80x9d seeds based on encapsulation and/or coating of somatic embryos will have practical utility. However, it should be noted that the germination vigor of naked, i.e., un-coated or non-encapsulated somatic embryos produced with methods disclosed in the art and then sown using aseptic technique onto in vitro germination media and subsequently germinated in sterile conditions, can approximate those of the corresponding zygotic seeds (e.g., greater than 85%) (Gupta and Grob, 1995).
Timmis et. al. (U.S. Pat. No. 5,119,588 incorporated herein by reference) teach a method by which plant somatic embryos can be sown into horticultural containers filled with particulate soil-like substrates. Solutions containing carbon compounds serving as energy sources and other nutrients are added to the substrates before or after the embryos are sown. However, they teach that it is essential that the containers, substrate, nutrient solutions and other components of their system must be biologically sterile. Furthermore, once the somatic embryos are sown into their system using aseptic techniques, each individual container must be kept biologically separated from the others and from the external environment and must be kept in a sterile condition until the embryo has successfully germinated, formed functional roots and shoots and has accomplished autotrophy. Only after autotrophy has been reached can the somatic seedlings be removed from the sterile conditions within their system, and then transplanted into a conventional commercial propagation environment.
Even though it is possible to successfully germinate plant somatic embryos on culture media using aseptic techniques and subsequently transplant the germinants into nursery production environments, such methods are labor-intensive, slow and very costly. Furthermore, somatic embryos obtained in accordance with the prior art are not amenable to the systems and equipment commonly used for commercial production of plant material. Therefore, there is still a need for a practical method of supplying exogenous energy and nutrient sources to plant somatic embryos in a manner that will facilitate and maximize the ex vitro germination of somatic embryos and the subsequent plant development and growth under non-sterile conditions.
An object of the invention is to enhance germination of plant somatic embryos.
Another object of the invention is to improve the reliability and synchronization of plant somatic embryo germination.
Another object of the invention is to facilitate the growth of plants and seedlings from plant somatic embryos.
Another object of the invention is to enable the germination of plant somatic embryos to occur in non-sterile conditions in growing media commonly used in commercial nursery practice.
According to one aspect of the invention, there is provided a process of priming plant somatic embryos, which comprises contacting mature plant somatic embryos with an aqueous solution containing a dissolved nutrient.
The nutrient is preferably a carbohydrate, e.g. a sugar such as sucrose. When sucrose is employed as the priming nutrient, it is preferably used at a concentration in an aqueous medium of 6% w/v or less.
According to another aspect of the invention, there is provided a process of nutripriming plant somatic embryos prior to germination, comprising contacting imbibed plant somatic embryos with a solution comprising a mixture of two or more dissolved nutrients.
According to another aspect of the invention, there is provided a method of producing seedlings or full-grown plants from somatic embryos, which comprises nutripriming plant somatic embryos by contacting imbibed plant somatic embryos with a solution containing a dissolved nutrient, germinating the nutriprimed embryos in a growth medium to form germinants, and maintaining growing conditions to allow the germinants to grow into seedlings or full-grown plants.
According to another aspect of the invention, there is provided a method of producing seedlings or full-grown plants from somatic embryos, which comprises nutripriming plant somatic embryos by contacting imbibed plant somatic embryos with a solution containing a mixture of dissolved nutrients, germinating the nutriprimed embryos in a growth medium to form germinants, and maintaining growing conditions to allow the germinants to grow into seedlings or full-grown plants.
According to another aspect of the invention, there is provided a method of germinating mature plant somatic embryos, comprising priming imbibed embryos in the presence of a nutrient medium.
According to yet another aspect of the invention, there is provided a method of producing somatic seedlings, comprising (i) priming imbibed mature plant somatic embryos in a nutrient medium, and (ii) sowing the embryos in a growth medium.
The invention also relates to somatic embryos, seedlings or mature plants produced by the above methods.
The somatic embryo is preferably from a tree species, most preferably a gymnosperm, e.g. pine. The process is particularly effective with spruce and pine somatic embryos.
It is an advantage of the present invention, at least in preferred forms, that it can provide a process by which an imbibed somatic embryo can be nutriprimed in solutions containing one or more nutrients, then harvested from the nutripriming medium, and subsequently sown, germinated and grown ex vitro using conventional horticultural and agricultural equipment, containers, growing substrates, and growing environments. Furthermore, the sowing, germination and growing steps can be performed without the use of biologically isolating enclosures, or sterile media and growing environments. Alternatively, after the nutripriming step is completed, primed somatic embryos can be dried and stored for periods of time prior to ex vitro sowing and germination
Another advantage of the invention, at least in preferred forms, is that it can provide a process by which the nutripriming of somatic embryos followed by harvesting and subsequent ex vitro sowing, germination and growing, can be practiced with a diverse variety of gymnosperm and angiosperm species. Alternatively, harvested nutriprimed somatic embryos of both gymnosperm and angiosperm species may be desiccated and stored for periods of time prior to ex vitro sowing and germination.
In preferred forms, the present invention relates to a multi-step process to produce seedlings from mature somatic embryos which begins by imbibing somatic embryos and then placing the imbibed somatic embryos into solutions containing nutrients for periods of time. This first component of the multi-step process is referred to as xe2x80x9cnutripriming.xe2x80x9d It has surprisingly been found that nutriprimed somatic embryos can be sown ex vitro using various non-sterile methods into a wide variety of horticultural nursery containers filled with various types of non-sterile growing mixes commonly used in commercial horticultural and agricultural plant propagation. Once placed into conventional non-sterile nursery propagation systems and using conventional horticultural growing practices, the nutriprimed somatic embryos will germinate and grow into fully functional plants. Furthermore, we have surprisingly found that nutripriming solutions can be applied using conventional non-sterile horticultural equipment to facilitate and enhance the germination of nutriprimed somatic embryos.
We have also surprisingly discovered that nutriprimed somatic embryos can be desiccated to moisture contents in the range of 10-76%. Furthermore, we have discovered that desiccated nutriprimed somatic embryos can be stored for extended periods of time without significant declines in physiological integrity or germination potential. We have also discovered that desiccated nutriprimed somatic embryos are amenable for sowing with conventional seeding equipment into conventional plant propagation media for germination and further growth and development using conventional non-sterile plant propagation practices.
Consequently, the multi-step process of at least preferred forms of the present invention includes, but is not limited to, the steps of imbibing somatic embryos, nutripriming said imbibed somatic embryos, placing said nutriprimed somatic embryos into a state of physiological dormancy, sowing said nutriprimed physiologically dormant somatic embryos onto or into conventional horticultural germination substrates, propagating said sown nutriprimed somatic embryos in environmental conditions manipulated to facilitated imbibition, germination, and development into complete seedlings possessing shoots and roots.
There are several advantages inherent with the use of the process of the invention. For example, one advantage of nutripriming plant somatic embryos is that they show exceptional vigor during germination and subsequent development into complete seedlings possessing shoots and roots. Furthermore, desiccated nutriprimed somatic embryos are particularly useful for preserving the physiological viability of the embryos during extended storage prior to sowing and germination. Yet another advantage of nutripriming somatic embryos is that they can be sorted according to size, length and shape to facilitate production of more uniform crops after sowing, germination and growth.
A key advantage of the present process is that once the nutripriming step is completed, all subsequent components of the multi-step process can be practiced in conventional plant propagation environments without the need for aseptic handling processes or for sterile growing environments. More specifically, aseptic procedures, and sterile or sanitized equipment and germination/growing environments are not required for successful ex vitro sowing and. germination of nutriprimed somatic embryos and their subsequent development into complete functional seedlings, thus enabling the entire sowing, germination, and growing steps to be performed, if so desired, in commercial plant propagation or greenhouse or nursery growing facilities.
Another advantage is that the nutriprimed somatic embryos can be sown with conventional seeding equipment such as but not restricted to, vacuum-drum seeders, fluid-drill seeders or needle-jet seeders.
A further advantage is that commonly used horticultural and agricultural products such as, but not restricted to, soil-less seedling mixes or rock wool or foams, can be used as the supports onto which the nutriprimed somatic embryos are sown and subsequently germinate into and penetrate with their roots.
Most preferably, a nutrient (ideally the same nutrient as the one used in the nutripriming step) is also incorporated into said growth medium.
Yet a further advantage is that if necessitated by the conditions in the commercial growing environments, existing commercial pesticide products such as, but not restricted to fungicides, bactericides, antibiotics, nematicides, insecticides and the like, which are registered for use with the plant species from which the somatic embryos are produced, can be applied to the sown nutriprimed somatic embryos per label instructions for effective pest control, or alternatively, applied to the growing substrates prior to sowing the somatic embryos.
Another advantage is that exogenous nutrients necessary for successful somatic embryo germination and growth can be applied via the various numerous methods commonly used in commercial horticulture, said methods including but not restricted to misting, fogging, spraying, watering and drenching. Furthermore, said exogenous nutrients can be applied in conjunction with conventional horticultural fertigation practices.
This invention includes the above objects and features taken alone and in combination. These and other features, objects and advantages of the present invention will become more apparent with reference to the following description.
A number of terms are known to have differing meanings when used in literature describing this art. The following definitions are believed to be ones most generally used in the fields of botany, plant somatic embryogenesis, and are consistent with the usage of the terms in the present specification.
These definitions will assist in the understanding of this detailed description.
xe2x80x9cABAxe2x80x9d is absicic acid, a plant growth regulator.
xe2x80x9cAutotrophicxe2x80x9d refers to the stage of plant development when the photosynthetic organelles and related enzymes and biochemical pathways are fully functional and capable of converting light energy, atmospheric carbon dioxide and water into the pre-requisite carbohydrates (e.g., glucose) necessary to sustain further plant growth and development.
xe2x80x9cBAxe2x80x9d is benzyl adenine, a cytokinin-type of plant growth regulator. The main physiological effect of BA is to stimulate meristomatic cell division.
xe2x80x9cClonexe2x80x9d when used in the context of plant propagation refers to a collection of individuals having the same genetic constitution, and are produced from a culture that arises from an individual explant.
xe2x80x9cEmbryogenic culturexe2x80x9d is a plant cell or tissue culture capable of forming somatic embryos and regenerating plants via somatic embryogenesis.
xe2x80x9cEndospermxe2x80x9d is haploid nutritive tissue of angiosperm seed, of maternal origin, within which the angiosperm zygotic embryos develop.
xe2x80x9cExplantxe2x80x9d is the organ, tissue or cells derived from a plant and cultured in vitro for the purposes of starting a plant cell or tissue culture.
xe2x80x9cGAxe2x80x9d is gibberellin, a group of related growth regulator isomers (e.g., GA3, GA4, GA7) that is naturally synthesized as a normal part of plant metabolism. The main physiological effect of GA is to stimulate elongation of individual plant cells. Exogenous applications of GA can be used to stimulate, manipulate and accelerate the initiation and growth of shoots and roots.
xe2x80x9cGerminationxe2x80x9d is the three-phase series of events which commences with the uptake of water by a quiescent dry seed and proceeds through various biophysical, biochemical and physiological events which result in the elongation of the embryo along its axis and ultimately concludes with the protuberance of a root or shoot radicle through the seed coat. Seed germination occurs in three phases. Phase one is characterized by a rapid initial influx of water into the seed accompanied by the initiation of repair to damaged DNA and mitochondria. Phase two is characterized by a significant reduction in the rate of water uptake accompanied by activation or de novo synthesis of enzymes that specialize in hydrolyzing the complex storage reserves of carbohydrates, proteins, and lipids in the embryo and the cotyledons or megagametophytes. The hydrolysis of these complex storage reserves during Phase two provides the substrates required for the respiration and growth of the zygotic embryos. Phase three is charactized by a second rapid increase in the rate of water uptake which is used primarily for the initiation of meristomatic cell division at the root and shoot apices of the embryo, and for uptake into the cells along the embryonal axis. The process of germination is complete when the embryo has elongated to the point of protrusion through the seed coat.
xe2x80x9cHydroprimingxe2x80x9d is a seed priming process by which seeds are misted or soaked in water and then dried back before they complete the germination process.
xe2x80x9cIAAxe2x80x9d is indole-acetic-acid, a auxin-type growth regulator naturally synthesized as normal part of plant metabolism. The main physiological effect of IAA is to stimulate meristomatic cell division. Exogenous applications of IAA can be used to stimulate, manipulate and accelerate the initiation and growth of shoots and roots.
xe2x80x9cIBAxe2x80x9d is indole-butyric-acid, a chemical analog of IAA. IBA can be used to affect the initiation of roots and shoots in the same manner as exogenous applications of IAA.
xe2x80x9cImbibitionxe2x80x9d is the absorption and/or adsorption of water by certain colloids present in seeds or embryos, which results in the swelling of the tissues and activation of enzymatic and physiological processes.
xe2x80x9cLinexe2x80x9d is another term for xe2x80x9cclonexe2x80x9d.
xe2x80x9cMatriprimingxe2x80x9d refers to the use of solid carriers with low matric water potentials to control the rate and/or the amount of water absorbed during a seed priming process.
xe2x80x9cMegagametophytexe2x80x9d is haploid nutritive tissue of gymnosperm seed, of maternal origin, within which the gymnosperm zygotic embryos develop.
xe2x80x9cMicrodropletxe2x80x9d is small molecule of water or water-based solution contained within the fine spray produced by applying pressure to a drop of water or a water-based solution.
xe2x80x9cNutrientsxe2x80x9d are the inorganic micro- and macro-minerals, vitamins, hormones, organic supplements, and carbohydrates necessary for culture growth and somatic embryo germination.
xe2x80x9cNutrient solutionxe2x80x9d is water containing a dissolved nutrient or mixture of nutrients.
xe2x80x9cNutriprimingxe2x80x9d refers to a process of exposing a plant somatic embryo to a nutrient solution during the Phase two period of germination for a period of time sufficient to enable the embryo to absorb mineral and organic nutrients necessary to complete the Phase two and Phase three steps of germination.
xe2x80x9cOsmoprimingxe2x80x9d refers to a method of soaking seeds in aerated osmotica of low water potential to control the rate and/or the amount of water absorbed during a seed priming process.
xe2x80x9cPhysiological dormancyxe2x80x9d refers to the cessation of the normal metabolic processes, i.e., anabolism and catabolism, that are inherent in plant growth and development, in a manner that does not negatively affect viability.
xe2x80x9cPregerminationxe2x80x9d is a seed priming process which allows water availability to seeds to the point of shoot or root radicle protrusion from the seed coat, before the desiccation step is applied.
xe2x80x9cSeed primingxe2x80x9d refers to a process which controls and manipulates the water availability to seeds to initiate and affect the early events of germination, but not sufficient to permit radicle protrusion, which is subsequently followed by drying.
xe2x80x9cSomatic embryogenesisxe2x80x9d is the process of initiation and development of embryos in vitro from somatic cells and tissues.
xe2x80x9cSomatic embryoxe2x80x9d is an embryo formed in vitro from vegetative (somatic) cells by mitotic division of cells. Early stage somatic embryos are morphologically similar to immature zygotic embryos; a region of embryonal cells subtended by elongated suspensor cells. The embryonal cells develop into the mature somatic embryo.
xe2x80x9cZygotic embryoxe2x80x9d is an embryo derived from the sexual fusion of gametic cells.
In a preferred form, the present invention is generally a multi-step germination process for plant somatic embryos which enables the use of conventional horticultural practices, equipment and facilities, said process comprising but not restricted to, some or all of the following sequential steps:
1. Initiating Phase one of the germination process by imbibing desiccated mature plant somatic embryos. It is preferable that the rate of imbibition during Phase one is controlled through the use of a process such as matripriming. It is also preferable that this step is performed using aseptic techniques and sterile conditions.
2. Once the Phase one germination step, i.e., imbibition, has been completed, the imbibed plant somatic embryos are transferred to a vessel containing a liquid nutripriming solution. The nutripriming solution must contain at least one source of carbohydrate. Although the preferred carbohydrate is sucrose, preferably in the range of 3-6% (w/v), this invention can be practiced with sugars such as fructose, glucose, maltose, galactose, mannose, lactose and the like. Furthermore, the nutripriming solution may contain, if so desired, a mixture of two or more carbohydrates. If carbohydrates other than sucrose, or if mixtures of carbohydrates, are used in the nutripriming solution, then the appropriate concentrations of each carbohydrate should be determined in advance by the use of rate-selection studies. The design and performance of such rate-selection studies are known to those skilled in this art. Another key feature of the present invention is that in addition to carbohydrates, the nutripriming solutions may also contain, if so desired, other types of nutrients which may further facilitate the various biochemical and physiological processes occurring during germination Phases two and three. Such nutrients include but are not restricted to inorganic minerals, vitamins and hormones. A non-limiting example of how this can be practiced is by adding to a solution containing sucrose in the range of 3-6% (w/v), a mixture of mineral nutrients formulated to deliver but not restricted to 454 mg/l nitrogen, 81 mg/l phosphorus, 704 mg/l potassium, 50 mg/l calcium, 39 mg/l magnesium, 193 mg/l sulfur, 3 mg/l manganese, 0.5 mg/l zinc, 89 mg/l chlorine, 3 mg/l iron, 0.7 mg/l iodine, 0.6 mg/l boron, 0.01 mg/l molybdenum, 0.01 mg/l cobalt, and 0.01 mg/l copper. Furthermore if so desired, IBA a plant growth regulator, may be added alone at a concentration of 0.1 uM/l or in combination with one or both of GA and BA, each at a concentration of 0.1 uM/l. Also, Ascorbic acid (a.k.a. vitamin C) may be added if so desired, at a concentration in the range of 10-1000 uM/l. Furthermore, if so desired, pest control products such as antibiotics or fungicides may be added to the nutripriming solution. A non-limiting example is the addition of benlate (0.1 g/l) and/or ampinicillin (0.1 g/l). It is preferable during this step that the nutripriming solutions are sterilized prior to addition of the somatic embryos, and that aseptic technique is used when adding embryos to the nutripriming solution.
3. The imbibed embryos are nutriprimed in the nutripriming solution for a period of time preferably ranging from 6 hours to 168 hours, more preferably in the range of 48 to 96 hours and most preferably between 72 to 96 hours. It is also preferable throughout the nutripriming process, that the embryos are suspended within the nutripriming solutions and are kept in constant motion. A non-limiting example of how this might be accomplished is by securing the vessels containing the embryos and nutripriming solutions onto a shaker table which is revolving at a rate in the range of 10-120 rpm, preferably in the range of 30-60 rpm. It is possible to practice the nutripriming step in either the presence or absence of light. Since it is known in the art that zygotic seeds of certain plant species will germinate only in the dark while zygotic seeds of other plant species require light for successful germination, those skilled in the art will be able to determine if the nutripriming step should be illuminated for the plant somatic embryos with which they wish to practice this invention. It is preferable that the contents of the nutripriming vessels are maintained in a sterile condition during the nutripriming process.
4. Sowing the nutriprimed plant somatic embryos into nursery containers containing a three-phase conventional horticultural growing substrate, said three phases comprising solids, liquids and air. Commencing with this step, aseptic technique and sterile conditions are not required to successfully practice this invention.
5. Placing the nursery containers sown with nutriprimed plant somatic embryos, into a conventional plant propagation environment in which light, temperature, atmospheric humidity, and moisture content of the rooting substrate can be controlled and manipulated to enable and facilitate the re-germination of the somatic embryos and their further development into seedlings.
6. If so desired, supplying an aerosol in the form of a mist or spray, to the surface of the nursery containers sown with somatic embryos, said aerosol containing the necessary carbohydrate compounds required to sustain and facilitate completion of the Phase three germination processes of the somatic embryos.
7. Supplying in the forms of an aerosol and/or a liquid suspension and/or a liquid solution, the micro- and macro-mineral elements required to sustain and facilitate completion of the Phase three germination processes of the somatic embryos and their subsequent development into seedlings.
8. Adjusting as required during completion of the somatic embryo germination processes and subsequent development into seedlings, the ambient light intensity and diurnal photoperiod, temperature, atmospheric humidity and other such factors may be adjusted as required during somatic embryo germination and their conversion into fully functional seedlings.
Alternatively, at the completion of step 3, nutriprimed embryos may be removed from the nutripriming solutions and desiccated to a moisture content preferably in the range of 10% to 75%, more preferably in the range of 15% to 50% and most preferably in the range of 20% to 25%. For example, the embryos may be desiccated by pouring the nutripriming solutions with the nutriprimed embryos onto filter paper in a vacuum filter apparatus for the removal of the excess solution; other methods of desiccation are known to those skilled in the art. The filter paper with primed embryos on their surfaces can then be placed into xe2x80x9cflow-throughxe2x80x9d desiccation chamber for a period of 3-96 hr, preferably 12-24 hr. After desiccation, the dried nutriprimed embryos can be stored in sealed, plastic-lined pouches. Although desiccated nutriprimed embryos can be stored at ambient temperatures, it is preferable that they are stored at refrigerated temperatures, e.g., 2 to 10xc2x0 C., and most preferably, frozen e,g, xe2x88x9220 to xe2x88x9280xc2x0 C. Although it is preferable to use aseptic technique during the desiccation and storage of nutriprimed embryos, it is not essential for the successful practice of this invention. Desiccated nutriprimed embryos can be removed from storage and sown ex vitro commencing with step 4 above.
A key feature of this novel process is that special hygenic and/or aseptic and/or sterile handling methods and/or equipment and/or facilities are not required to successfully handle, sow and germinate wet or desiccated nutriprimed plant somatic embryos. Accordingly, steps 4 through 8 may be carried out in non-sterile, unhygenic and/or septic conditions, i.e., those types of conditions typically encountered in conventional horticultural plant propagation environments.
Although the nutriprimed somatic embryos can be sown with all types of conventional seeding equipment used for sowing zygotic seeds, it is preferred to use equipment that dispenses singulated seed into multi-chambered nursery containers, commonly referred to as miniplug trays or cell-packs, said containers commonly used to produce plant plugs which can be mechanically transplanted into larger containers or into field-growing environments.
A key feature of the present invention, at least in its preferred forms, is that the sowing and propagation of nutriprimed somatic embryos can be practiced with a wide variety of non-sterilized growing substrates commonly used in conventional plant propagation. The preferred growing substrate is peat-based and has been formulated specifically for germination of zygotic seed and is exemplified by mixtures such as (a) 100% short-fiber peat product polymerized with a water-binding polymer, supplied by companies such as Grow Tech Inc. (San Juan Bautista, Calif. USA), Preforma Inc. (Oberlin, Ohio USA), (b) 15.2 cu.ft of peat, 8 cu.ft. of vermiculite, 680 grams of dolomite lime, and 300 grams of Micromax(copyright), and (c) 16.2 cu.ft. of peat, 6.75 cu.ft. perlite, 4 cu.ft. vermiculite, 6 kilograms of dolomite lime, 1.5 kilograms of gypsum, 375 grams of potassium phosphate, 250 grams Micromax(copyright), and 35 grams of wetting agent. Alternatively, commercially formulated mixes such as PRO-MIX-G(copyright) or PRO-MIX-PGX(copyright) (Premier Peat Moss Ltd. Montreal, PQ, Canada), Sunshine Mix #3 (Sun-Gro Horticulture Inc., Hubbard, Oreg., USA), and Redi-Earth(copyright) (The Scotts Co., Marysville, Ohio, USA) can also be used with the present invention. It is preferred that the peat-based growing substrate is moistened to a moisture content in the range of 59-75% and then dispensed into multi-chambered trays commonly used for production of plant plugs. Although examples of such trays include styrofoam #252 miniplug trays manufactured by Beaver Plastics Inc (Edmonton, AB, Canada) and hard plastic #288 or #512 miniplug trays manufactured by TLC Polyform Inc (Plymouth Minn., USA, 55441), the present invention can be practiced with other such multi-chambered trays, or alternatively, with individual pots. It should be noted that the practice of the present invention is not restricted to peat-based mixtures, but also includes other substrate such as Jiffy-7 peat plugs, composted or shredded coconut husk fibres commonly referred to as xe2x80x9ccorxe2x80x9d or xe2x80x9ccoirxe2x80x9d (1993 Crystal Co., St. Louis, Mo., USA), extruded foams such as Oasis(copyright) (Smithers-Oasis Ltd., Kent, Ohio, USA), rock wool (Rockwool International A/S, Hovedgaden 584, DK-2640, Denmark) and the like. Regardless of the rooting substrate chosen, its physical characteristics should enable development and maintenance of a high relative humidity in the gaseous phase, i.e., in excess of 75% RH, within the substrate while minimizing saturation of the substrate with the liquid phase.
After the pre-germinated somatic embryos are sown onto the surfaces of the rooting substrates, if so desired, the embryos may be covered with a thin layer of additional rooting substrate that may be comprised of the same material underneath the embryos or alternatively, with a different type of material. One non-limiting example is sowing the pre-germinated embryos onto PRO-MIX-PGX(copyright) medium, then overlaying the embryos with a thin layer of coconut husk fibres.
Nursery containers sown with pre-germinated somatic embryos are preferentially placed into a conventional plant propagation environment wherein the conditions are within but not limited to the ranges of temperatures of 15-35xc2x0 C., relative humidities of 75-100%, light intensities of 10-500 foot candles, and diurnal cycles of 6 h day/18 h night-22 h day/2 h night.
It is preferable to maintain a very high level of atmospheric humidity around the nursery containers sown with pre-germinated somatic embryos, i.e., greater than 90% RH, for the first 3-7 days after sowing to facilitate somatic embryo imbibition and germination. A number of methods can be used to maintain the atmospheric humidity at these levels including but not restricted to placing the containers in a greenhouse environment with misting or fogging equipment which is deployed at controlled intervals, placing the containers in a fogging or misting tent or chamber, placing clear plastic domes over the nursery containers and then removing domes periodically to mist or fog the sown embryos and replacing the domes immediately thereafter. Another non-limiting method is to provide a space ranging between 2 mm and 10 mm above the surface of the rooting substrate onto which the embryos are sown and the top of the container, and then covering the top of the nursery container with a plastic film which is removed to enable misting or fogging of the sown embryos and then immediately replaced. After somatic embryo germination is established as evidenced by development of epicotyl and root structures, the germinants can be weaned from the high relative humidity environments and integrated into conventional nursery cultural practices by gradually reducing the amount of misting/fogging applied and/or by extending the periods of time between the misting or fogging steps.
It is preferable to maintain the sown pre-germinated embryos in a high relative humidity environment, i.e., greater than 90% RH, for a period of, but not restricted to, 3-7 days after sowing to facilitate embryo imbibition, prior to supplying exogenous nutrients required for embryo germination.
Another key feature of the invention, at least in its preferred forms, is that the exogenous nutrients, including but not restricted to carbohydrates and minerals, required for successful somatic embryo re-germination and subsequent growth and development can be applied as aerosols. The nutrient solutions can be applied with, but not restricted to, conventional misting and/or fogging equipment. Although, the nutrients can be applied individually or combined into one solution, it is preferred to supply the carbohydrates as one solution and the remaining nutrients as a separate solution. A non-limiting example of how this can be practiced is by applying a 3% w/v sucrose solution as a mist to the surface of the growing substrate containing a sown pre-germinated embryo, and then applying at a later time, a solution containing a mixture of mineral nutrients formulated to deliver 454 mg/l nitrogen, 81 mg/l phosphorus, 704 mg/l potassium, 50 mg/l calcium, 39 mg/l magnesium, 193 mg/l sulfur, 3 mg/l manganese, 0.5 mg/l zinc, 89 mg/l chlorine, 3 mg/l iron, 0.7 mg/l iodine, 0.6 mg/l boron, 0.01 mg/l molybdenum, 0.01 mg/l cobalt, and 0.01 mg/l copper. Alternatively, the macronutrients can be supplied as a commercial formulation such as but not restricted to PlantProd(copyright) Plant Starter Fertilizer 10-52-10 (nitrogen-phosphate-potassium) or PlantProd(copyright) Forestry Seedling Starter 11-41-8 (nitrogen-phosphate-potassium) (Plant Products Ltd., Brampton, ON, Canada).
An alternative non-limited means of supplying exogenous nutrients to pre-germinated somatic embryos sown onto three-phase growing media within nursery containers is to irrigate or xe2x80x9cdrenchxe2x80x9d the media with nutrient solutions formulated as previously described.
Since microorganisms such as fungi, bacteria, yeast, and algae, are ubiquitous in conventional plant propagation substrates, equipment, containers and growing environments, a wide variety of chemical and biological pesticide products are available to control and irradicate plant pathogens. It has surprisingly been found that aseptic handling procedures and sterilized growing substrates, nursery containers and environments are not required to successfully germinate and grow plant somatic embryos. Indeed, the invention can be practiced in conventional plant propagation environments using only the standard commercial methods of hygiene. Furthermore, we have surprisingly found that pesticides such as Benlate(copyright), Rovril(copyright), Trumpet(copyright) and the like, which registered for pest control in plant crops, can be used in conjunction with somatic embryos pre-germinated and subsequently sown with the present novel multi-step procedure.